Effective macrophage clearance of Klebsiella pneumoniae requires the inducible nitric oxide synthase iNOS and is independent of reactive oxygen species generated by NADPH oxidase

Klebsiella pneumoniae is a leading cause of pneumonia and bacteremia and is especially dangerous in healthcare settings. Despite massive clinical significance, the mechanisms used by macrophages to kill K. pneumoniae are largely unknown. Macrophages are critical for controlling K. pneumoniae, as mice lacking monocyte-derived or alveolar macrophages have higher bacterial tissue burdens and mortality. Two prominent mechanisms used by macrophages to kill bacteria are the production of reactive oxygen species (ROS) via the NADPH oxidase NOX2 and reactive nitrogen species (RNS) via the inducible nitric oxide synthase iNOS. Previously, we found that a subset of K. pneumoniae factors which enhance bacteremia pathogenesis also support intracellular survival. The degree to which these factors enhance intracellular fitness correlated with resistance against RNS, not ROS. Here, we aimed to define the extent to which macrophage ROS and RNS contribute to K. pneumoniae clearance. Using wild-type, Cybb-/-, and Nos2-/- cells, we measured K. pneumoniae survival in macrophages lacking such defenses. NOX2 was dispensable for K. pneumoniae elimination, and ROS was undetectable in K. pneumoniae-infected macrophages. We confirmed that ROS was also undetectable within alveolar-like macrophages, indicating that ROS is dispensable for anti-K. pneumoniae defense across macrophage subsets. Instead, iNOS contributed to macrophage clearance of K. pneumoniae and likely enhances killing by RNS induction and coordinating a normal cytokine response. Activation of pathways upstream of iNOS may be the most relevant to supporting effective macrophage control of K. pneumoniae. This study defines unexpected differential roles for ROS and RNS in macrophage clearance of K. pneumoniae.